PROJECT SUMMARY The increasing prevalence of HIV-1 drug resistance (DR) is a threat to HIV-1 treatment and prevention in the low- and middle-income countries hardest hit by the HIV-1 pandemic. An inexpensive genotypic resistance test (GRT) that could be performed during a clinic visit, heretofore called a point-of-care (POC) assay, would enable HIV-1 care providers to make informed treatment decisions for antiretroviral (ARV)-nave patients starting therapy and for patients with virological failure (VF) on therapy. Through the delivery of immediately actionable information upon the detection of VF, a POC GRT would benefit treatment programs by reducing treatment delivery costs and would benefit patients by reducing travel costs and work absenteeism, and by increasing the likelihood treated patients achieve and maintain suppressed virus levels. An assay that detected the DR mutations (DRMs) K65R/N, K103N/S, V106A/M, Y181C/I/V, M184V/I and G190A/S would be highly sensitive for detecting acquired DR on an NRTI/NNRTI-containing regimen and for detecting transmitted DR that would compromise a first-line NRTI/NNRTI-containing regimen. The main challenge in developing a POC GRT is the genetic variability at and surrounding each DRM position. We describe preliminary experiments using a novel sold-phase nucleic acid melt-analysis platform that demonstrate a high sensitivity and specificity for detecting multiple variants at the DRM position 103. We describe plans to collaborate with the biotechnology company InSilixa (Sunnyvale, CA) to develop the required solid-phase fluorogenic probe sets and a multiplex asymmetric RT PCR protocol to detect the 35 most common codons at the DRM positions 65, 103, 106, 181, 184, and 190. The probe sets and PCR protocol will be optimized and validated using a dynamic microarray setup predictive of probe performance on InSilixa?s complementary metal-oxide semiconductor (CMOS) biochip-based HYRDRA-1K POC platform. If the aims of this proposal are met, we will extend our collaboration with InSilixa to (i) evaluate sample processing modules to separate plasma from blood, concentrate virus, and extract RNA that can be delivered to the fluidic chamber of the HYDRA-1K POC platform; (ii) test a wide range of clinical samples; (iii) transfer the optimized probe sets onto the HYDRA-1K POC platform; (iv) determine whether the presence of probe hybridization can be used to define a reproducible threshold for detecting HIV-1; and (v) extend the assay to include protease inhibitor and integrase inhibitor DRMs. Developing a POC HIV-1 GRT will also address the broader challenge of detecting mutations in rapidly evolving epidemic viruses whether they are DRMs or critical vaccine-escape or gain-of-function mutations.